WO2002033392A2

WO2002033392A2 - Electrophoresis tank

Info

Publication number: WO2002033392A2
Application number: PCT/US2001/024911
Authority: WO
Inventors: N. Leigh Anderson; Jack Goodman
Original assignee: Large Scale Proteomics Corporation
Priority date: 2000-08-09
Filing date: 2001-08-09
Publication date: 2002-04-25
Also published as: AU2001283204A1; WO2002033392A3

Abstract

An electrophoresis tank method and apparatus is provided. The electrophoresis tank comprises an outer shell, a plurality of receptacles attached to a bottom of the tank, the plurality of receptacles being adapted to receive a plurality of sealing strips and being formed in two substantially parallel rows and dividing the tank into a first region, a second region, and a third region, with the third region being disposed between the first region and the second region,and a first electrode at a first side of the tank and a second electrode at a second side of the tank, with a first electrode being entirely within the first region and the second electrode being entirely within the second region, wherein in use a plurality of electrophoresis gel slabs are inserted in between the plurality of sealing strips so that a first end of a gel slab is positioned in the first region and a second end of the gel slab is positioned in the second region and a main body of the gel slab extends through the third region, and wherein when the tank is filled with the plurality of gel slabs, the plurality of sealing strips contact the plurality of gel slabs to form the third region.

Description

ELECTROPHORESIS TANK

5 BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to an electrophoresis apparatus.

2. Description of the Background Art ₀ Gel electrophoresis is a process for distinguishing and identifying organic macromolecules . Some of the uses of gel electrophoresis include protein analysis and DNA analysis. Gel electrophoresis typically separates macromolecule components in one or two dimensions to provide a result 5 wherein individual macromolecule components appear as bands or spots. The resulting bands or spots may be further analyzed.

A wide variety of processes and equipment have been developed to improve the gel electrophoresis process through large-scale automation. o Gel electrophoresis gets its name from a polyacrylamide gel used in the process. Sample macromolecules to be analyzed are embedded in the gel . The gel and embedded macromolecules are electrophoresed (subjected to an electric field) to produce the physical separation of the component 5 macromolecules .

In a first dimension electrophoresis, a test sample is applied to the gel and electrophoresed. As a result of the electric field, the macromolecule components in the test sample migrate and become physically separated in one dimension on the basis of their electrical charges.

In the second dimension electrophoresis, the product of the first dimension electrophoresis is further separated on the basis of molecular weight.

The gel slab is typically electrophoresed in a tank holding a buffer fluid. The buffer fluid conducts electricity, with the purpose of the buffer fluid being primarily to form a complete circuit from a first electrode, to a first end of a gel slab (or gel cassette) , through the electrophoresis gel, and then from a second end of the gel slab to a second electrode. The gel slab or slabs are therefore desired to be in the path of an electrical current flow between the electrodes, and that the electrical current flow not circumvent the gel slabs. In order to accomplish this, the electrophoresis tank should be constructed so that the buffer fluid in the regions at the ends of the gel slabs are isolated from each other (i.e., fluid tight) .

Related art electrophoresis apparatus has typically suffered from several drawbacks. First, there has been the problem of fluid leakage between the fluid in the electrode regions and the region between the gel slabs . This can cause electrical current leakage and an uneven electrophoresis effect. Second, an electrophoresis apparatus will experience some degree of heating, and related art apparatus often do not provide temperature compensation. As a result, the buffer fluid and the gel slabs may contain regions of varying temperatures, so-called "hot spots" . Temperature variations in the buffer fluid or in the gel slabs may cause an uneven electrophoresis effect. Third, the related art may not adequately circulate the buffer fluid, potentially causing both hot spots and depletion of ions in the buffer fluid. Because the electrical current flow through the buffer fluid is impeded by this ion depletion, there is a tendency of the electrophoresis process to slow down over time.

Fourth, generally all electrophoresis operations are controlled by an operator, and may therefore suffer from imprecision or a lack of attention. If the operator does not monitor the temperature closely, the electrophoresis tank may be over or under cooled, leading to an uneven or otherwise unsatisfactory electrophoresis result. If the operator does not stop the electrophoresis process at an optimum time, the result may not be satisfactory. Likewise, if the operator does not adequately control the circulation of the buffer fluid, the temperature and ion distribution of the fluid may be less than adequate, leading to a decreased or sub optimal result.

Finally, because the related art devices are not computer controlled, they fail to adequately streamline and speed the electrophoresis process. Automation of the process can allow increased throughput and efficiency. There remains a need in the art, therefore, for improvements in electrophoresis processing apparatus.

SUMMARY OF THE INVENTION An electrophoresis tank adapted for use with a plurality of electrophoresis gel slabs is provided according to a first aspect of the invention. The electrophoresis tank comprises an outer shell forming an electrophoresis compartment capable of containing a buffer fluid, a plurality of receptacles attached to a bottom of the tank, the plurality of receptacles being adapted to receive a plurality of sealing strips and being formed in two substantially parallel rows and dividing the tank into a first region, a second region, and a third region, with the third region being disposed between the first region and the second region, and a first electrode at a first side of the tank and a second electrode at a second side of the tank, with the first electrode being entirely within the first region and the second electrode being entirely within the second region, wherein in use a plurality of electrophoresis gel slabs are inserted in between the plurality of sealing strips so that a first end of a gel slab is positioned in the first region and a second end of the gel slab is positioned in the second region and a main body of the gel slab extends through the third region, and wherein when the tank is filled with the plurality of gel slabs, the plurality of sealing strips contact the plurality of gel slabs to form the third region, with the third region being substantially fluid tight and electrically insulated from the first region, the second region, and the third region. An electrophoresis tank adapted for use with a plurality of electrophoresis gel slabs is provided according to a second aspect of the invention. The electrophoresis tank comprises an outer shell forming an electrophoresis compartment capable of containing a buffer fluid, a fluid inlet located on the outer shell, a fluid outlet located on the outer shell, a plurality of receptacles attached to a bottom of the tank, the plurality of receptacles being adapted to receive a plurality of sealing strips and being formed in two substantially parallel rows and dividing the tank into a first region, a second region, and a third region, with the third region being disposed between the first region and the second region, a first electrode at a first side of the tank and a second electrode at a second side of the tank, with the first electrode being entirely within the first region and the second electrode being entirely within the second region, three temperature sensors, the three temperature sensors being located in the first region, the second region, and the third region, three cooling devices, the three cooling devices being located in the first region, the second region, and the third region, a fluid level sensor located in the first or second regions, two fluid circulating devices, the two fluid circulating devices being located in the first region and the second region, and a computer in communication with the three temperature sensors, the three cooling devices, the fluid level sensor, and the two fluid circulating devices, the computer being capable of filling the electrophoresis tank with a buffer fluid, equalizing a buffer fluid level within the electrophoresis tank after insertion of a plurality of electrophoresis gel slabs, placing a predetermined voltage potential across the first and second electrodes to start electrophoresis, monitoring temperature level signals from the three temperature sensors, controlling the three cooling devices according to a corresponding temperature level signal, circulating the buffer fluid using the two fluid circulating devices, replenishing the buffer fluid in a region having a cathode electrode in order to maintain a predetermined ion supply level, and stopping electrophoresis after a predetermined time interval, wherein in use a plurality of electrophoresis gel slabs are inserted in between the plurality of sealing strips so that a first end of a gel is positioned in the first region and a second end of the gel slab is positioned in the second region and a main body of the gel extends through the third region, and wherein when the tank is filled with the plurality of gels, the plurality of sealing strips contact the plurality of gels to form the third region, with the third region being substantially fluid tight and electrically insulated from the first region and the second region.

An electrophoresis tank adapted for use with a plurality of electrophoresis gel slabs is provided according to a third aspect of the invention. The electrophoresis tank comprises an outer shell forming an electrophoresis compartment and including an open top, at least one positively chargeable electrode and at least one negatively chargeable electrode, a lid having an open position and a closed position wherein the lid closes the open top of the electrophoresis compartment, a lid sensor interposed between the lid and the outer shell, the lid sensor capable of detecting the open position of the lid, and a computer in communication with the lid sensor, the computer controlling a voltage and current applied to the at least one pair of electrodes to perform electrophoresis, wherein the computer terminates the voltage and the current when the lid sensor detects the open position of the lid.

A computer-implemented method for automating electrophoresis in an electrophoresis tank is provided according to a fourth aspect of the invention. The method comprises the steps of filling the electrophoresis tank to a predetermined level with a buffer fluid, equalizing a buffer fluid level within the electrophoresis tank after insertion of the plurality of electrophoresis gel slabs, placing a predetermined voltage potential across a first electrode and a second electrode to start electrophoresis, monitoring temperature level signals in each region of the three regions, controlling cooling devices in the each region according to a corresponding temperature level signal, circulating the buffer fluid in the each region, replenishing the buffer fluid in a region having a cathode electrode in order to maintain a predetermined ion supply level, and stopping electrophoresis after a predetermined electrophoresis time interval. The above and other features and advantages of the present invention will be further understood from the following description of the preferred embodiments thereof, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of an electrophoresis tank according to the present invention that is capable of holding a plurality of gel slabs and a volume of buffer fluid; FIG. 2 shows a top view of the electrophoresis tank of

FIG. 1;

FIG. 3 shows one embodiment of a sealing strip that may be used in the electrophoresis tank of the present invention; FIG. 4 shows an electrophoresis tank wherein a plurality of gel slabs have been installed between a plurality of sealing strips;

FIG. 5 shows electrophoresis devices of the electrophoresis tank;

FIG. 6 shows filling and buffer fluid level control devices of the electrophoresis tank;

FIG. 7 shows temperature control devices of the electrophoresis tank;

FIG. 8 shows buffer fluid circulation devices of the electrophoresis tank;

FIG. 9 is a perspective view of the tank showing lid, electrode, and outlet features;

FIG. 10 shows a portion of the electrophoresis tank, showing one embodiment of level control devices;

FIGS. 11 and 12 are cross-sections of first and second embodiments of the vertical tank walls having electrodes passing there through;

FIG. 13 shows a complete negative electrode and a complete positive electrode according to one embodiment of the present invention;

FIG. 14 is a partial cross-section, perspective view of the tank with one wall completely removed and a portion of another wall removed; FIG. 15 is similar to FIG. 14 but includes baffles; FIG. 16 is a cross-section of a bottom portion of the electrophoresis tank, showing the baffles;

FIG. 17 uses the cross-section to illustrate the flow of fluid due to the fluid circulating devices; and FIG. 18 shows a flowchart of a computer-implemented method of operating the electrophoresis tank.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a perspective view of an electrophoresis tank 100 capable of holding a plurality of gel slabs 110 and a volume of buffer fluid. The buffer fluid is used primarily to conduct electrical current from a pair of electrodes 142 to the plurality of gel slabs 110. As can be seen from the figure, the gel slabs 110 are not fully immersed in the buffer fluid 112. After the plurality of gel slabs 110 have been loaded into the electrophoresis tank 100, an electrical potential may be placed across the electrodes 142, so that the sample macromolecules in the gel slabs 110 will be spatially separated due to the second dimension electrophoresis process. The tank 100 in a preferred embodiment is constructed of a thick PLEXIGLAS, but could alternatively be constructed of any type of glass, plastic (such as, for example, LUCITE), or metal (treated to be electrically non-conductive) . The electrodes 142 are preferably formed of platinum, but alternatively may be formed of any other suitable electrically conductive metal .

FIG. 2 shows a top view of the electrophoresis tank 100. The electrophoresis tank 100 comprises an outer shell forming an electrophoresis compartment. The electrophoresis tank 100 includes at least one electrode pair 142 used to electrophorese samples. Also included in the electrophoresis tank 100 are a plurality of receptacles 123. The plurality of receptacles 123 approximately demarcate the electrophoresis tank 100 into a first region 101, a second region 102, and a third region 103. The third region 103 is disposed between the first region 101 and the second region 102. The receptacles 123 are included in the electrophoresis tank 100 to hold a plurality of sealing strips 302 (discussed below in conjunction with FIG. 3) .

FIG. 3 shows one embodiment of a sealing strip 302 that may be used in the electrophoresis tank 100 of the present invention. The sealing strip 302 has a height H, a substantially circular body 308, and flaps 309 extending from the body 308. A shaft 313 extends fully or partially through the body 308.

The flaps 309 may be substantially opposed. When inserted into the electrophoresis tank 100, the flaps 309 are displaced and deformed, forming essentially fluid tight seals between the gel slabs 110. The height H is preferably greater than or equal to a height of a gel slab 110.

The sealing strip 302 is preferably made of silicon rubber, but alternatively may be formed of any pliable material that is an electrical insulator.

The shaft 313 provides rigidity to the sealing strip 302, and is preferably glass reinforced plastic, although other materials may be employed. The sealing strip 303 may include an exposed shaft portion 316 that extends a predetermined distance below the body 308. This exposed shaft portion 316 is designed to fit into one of the receptacles 123 of the electrophoresis tank 100.

It should be understood that any type of sealing strip may be employed in the electrophoresis tank 100, and is not limited to those shown or described.

FIG. 4 shows an electrophoresis tank 100 wherein a plurality of gel slabs 110 have been installed between a plurality of sealing strips 302 extending upward from the bottom of the electrophoresis tank 100. As can be seen from the figure, the plurality of sealing strips 302 form an essentially fluid tight seal between the plurality of gel slabs 110. The result of installing the gel slabs 110 and sealing strips 302 is that the electrophoresis tank 100 is thereby divided up into the first region 101, the second region 102, and the third region 103. Therefore, the buffer fluid is segregated within each region (the gel slabs 110 are generally inserted into the electrophoresis tank 100 after the electrophoresis tank 100 has been filled with buffer fluid) . As a result, the ends of the electrophoresis gel 111 of each gel slab 110 are exposed to the first region 101 and the second region 102, and the electrical current is therefore constrained to flow essentially through the electrophoresis gel 111 without "short circuiting" through buffer fluid in region 103.

FIG. 5 shows electrophoresis devices of the electrophoresis tank 100. The electrophoresis tank 100 includes at least one pair of electrodes 142 connected to a computer 510. The computer 510 may control a voltage and current placed on the electrodes, and may additionally control a duration of the electrophoresis. The computer 510 may be any type of general purpose computer . The computer 510 preferably runs a LabVIEW software package, available from National Instruments, but any type of available process control software may be employed.

FIG. 6 shows filling and buffer fluid level control devices in the electrophoresis tank 100. The electrophoresis tank 100 includes a fluid inlet 636, at least one fluid outlet 602, a fluid level sensor 619, and an outlet valve 623. The electrophoresis tank 100 further includes a pump 633 connected to the tank inlet 636 by a conduit 638. The pump 633 is further connected to a buffer fluid reservoir 641 by a conduit 643. The computer 510 is connected by wires to the fluid level sensor 619. The fluid level sensor 619 provides a fluid level signal to the computer 510. By sensing the buffer fluid level in the electrophoresis tank 100 through use of the fluid level sensor 619, the computer 510 may control the addition or removal of buffer fluid. This may be done at various times, such as during an initial filling of the tank, during an equalization operation for equalizing the fluid level after insertion of gel slabs 110, during electrophoresis, such as in a replenishing operation, post- electrophoresis draining, or other instances. An alternate embodiment of the fluid level sensor 619 is discussed below in conjunction with FIG. 10.

The electrophoresis tank 100 can be operated with either a partial or full load of gel slabs. This capability is made possible through inclusion of the outlet 602. During a filling operation, the outlet 602 may allow excess buffer fluid to drain. The outlet 602 may be an open conduit set at a predetermined height in a tank sidewall, with the outlet 602 allowing the buffer fluid to fill only to that height. Alternatively, the outlet 602 may be a valve device that, under external control, opens to vent excess buffer fluid. The external control may be control by an operator or control by the computer 510 in conjunction with the fluid level sensor 619. The outlet 602 is further discussed below in conjunction with FIG. 9. During the replenishing operation, a predetermined volume of buffer fluid is removed through the outlet 602 and a desired volume of new buffer fluid is added. Replenishment may be done to ensure that the buffer fluid does not suffer from ion depletion.

The computer 510 may also control the removal of buffer fluid from the electrophoresis tank 100. The computer 510 may be connected by wires (not shown) to the outlet valve 623. Preferably the outlet valve 623 is located in a bottom region of the electrophoresis tank 100 so that draining is done by gravity. By opening the outlet valve 623, the computer 510 may allow the buffer fluid to drain from the electrophoresis tank 100. This may be done, for example, to equalize the buffer fluid level after insertion of gel slabs 110, or after electrophoresis is complete.

FIG. 7 shows temperature control devices of the electrophoresis tank 100. The electrophoresis tank 100 preferably includes three temperature sensors 714a- 714c and three cooling devices comprising, for example, cooling tubes 706a-706c and refrigeration units 708a-708c.

The computer 510 is connected by wires to the three temperature sensors 714a-714c. The temperature sensors 714a-714c are preferably thermocouples. The temperature sensors 714a- 714c provide the computer 510 with independent temperature level signals for the first region 101, the second region 102, and the third region 103. The cooling tubes 706a-706c and the refrigeration units 708a-708c are preferably conventional refrigeration devices or heat pumps, but other devices such as, for example, Peltier elements, etc., may be used. The refrigeration units 708a-708c typically provide a low temperature refrigerant to the cooling tubes 706a-706c. The cooling tubes 706a-706c absorb heat from the electrophoresis tank 100, and then the refrigerant is returned to the refrigeration units 708a-708c where the absorbed heat is removed.

A cooling tube 706 and a refrigeration unit 708 may comprise a refrigeration device available from GC Industries, for example.

The cooling tubes 706a-706c in the preferred embodiment run along the bottom of the electrophoresis tank 100 and in the inside thereof, but alternatively may be located on the underside of the electrophoresis tank 100 and absorb heat by conduction through the tank bottom.

The computer 510, responsive to temperature feedback from the temperature sensors 714a- 714c, is capable of controlling the flow of a low temperature coolant through the cooling tubes 706a-706c, with the computer 510 continuously or periodically receiving temperatures for each region. The low temperature coolant is preferably maintained at a temperature of about minus ten degrees Centigrade . By maintaining a desired temperature, the electrophoresis process may be improved. The temperature of the buffer fluid is therefore preferably maintained within a temperature range of about 10 degrees to about 20 degrees Centigrade. Even more preferably, the temperature is maintained at about 15 degrees Centigrade.

FIG. 8 shows buffer fluid circulation devices of the electrophoresis tank 100. The electrophoresis tank 100 includes one or more circulating devices, and preferably includes four circulating devices 804a- 804d connected by wires to the computer 510. One circulating device is located in the first region 101, one circulating device is located in the second region 102, and two circulating devices are located in the third region 103. The computer 510 is capable of turning on or off the one or more circulating devices 804a-804d, and is optionally capable of controlling and varying the speed of each circulating device. The circulating devices 804a-804d may be used to circulate the buffer fluid in each region. This circulation may evenly distribute the heat generated during electrophoresis, allowing a faster electrophoresis process. However, the main purpose of the circulating devices 804a- 804d is to force the buffer fluid past the cooling tubes 706a-706c to cool the buffer fluid (see FIG. 7 and FIGS. 15- 17 and accompanying text) . A circulating device 804 may be a motor turning an impeller. Alternatively, a circulating device 804 may be a motor and a corresponding pump driven by the motor. In either case, the computer 510 may include integral or independent motor controllers (not shown) .

In addition to heat distribution, the circulation provides an additional benefit of mixing the buffer fluid and buffer fluid additives. The buffer fluid may thereby be created in the tank 100 by adding the required amount of powder components into the tank 100 in a dry state, initiating automatic filling of a solvent (such as pure water) to a predetermined buffer fluid level, and then turning on one or more circulating devices. The resulting fluid circulation dissolves and mixes the dry and fluid contents to create the desired buffer fluid. Preferably, one or more sealing strips 302 are moved or removed during this mixing operation so that all three regions receive a completely mixed buffer fluid. The tank 100 preferably includes short extra slots (not shown) in the front and back walls to hold the removed sealing strips above the fluid level while the buffer fluid mixing occurs .

FIG. 9 is a perspective view of the tank 100 showing lid, electrode, and outlet features. The tank 100 may include a lid 904 that may be closed during an electrophoresis process to increase safety, as the electrophoresis process may operate at hundreds of volts. The lid 904 may optionally be affixed to the tank 100 by one or more hinges (not shown) , and may optionally include at least one latch 905. The at least one latch 905 may be used to removably affix the lid 904 to the tank 100 so that the lid 904 may not be accidentally opened during the electrophoresis process.

A lid sensor 907 may be interposed between the lid 904 and the outer shell of the tank 100. The lid sensor 907 therefore may be mounted either on the lid 904 or at or near the top of the tank 100. The lid sensor 907 may disable the electrophoresis electrodes when the lid 904 is not in place. The lid sensor 907 is preferably connected to an electrode power supply such that if the lid 904 is opened during the electrophoresis process, all electrical power to the electrodes 142 is cut off. Alternatively, the lid sensor 907 may be connected to the computer 510, and the computer 510 may control the electrode power supply.

The lid sensor 907 in a preferred embodiment is a magnetic switch, although alternatively the lid sensor 907 may be a mechanical switch (such as a simple contact controlling a relay), an optical switch, etc.

In a preferred embodiment, the outlet 602 (discussed in conjunction with FIG. 6) further comprises an upper outlet 912 and a lower outlet 913 that may be used to control a fill level of a buffer fluid. Preferably, the upper outlet 912 and the lower outlet 913 are located in the third region 103 (see FIG. 6) . In operation, the tank 100 is filled with a predetermined volume of buffer fluid. Depending on the number of gel slabs in the tank 100, a varying volume of buffer fluid will be displaced. The outlets 912 and 913 therefore maintain the buffer fluid level by discharging buffer fluid once the buffer fluid reaches the particular outlet 912 or 913.

Because the tank 100 may be used to electrophorese gel slabs of two different sizes, the upper outlet 912 and the lower outlet 913 may be used to accommodate either size of gel slab. For smaller gel slabs, the lower outlet 913 will maintain a lower fill level to accommodate a smaller gel slab. For larger gel slabs, the lower outlet 913 may be blocked (for example, plugged) , and as a result the upper outlet 912 will yield a higher fill level to accommodate the larger gel slab size.

It can also be seen from this figure that multiple positive and negative electrodes 142 and 143 may be positioned in the tank 100. The multiple electrodes create a- more even electric field than a single electrode pair and therefore a more even charge distribution across the two- dimensional gels during electrophoresis. In a preferred embodiment, the tank 100 includes twelve positive electrode pairs and twelve negative electrode pairs. It is also preferred that the negative and positive electrodes are symmetrically positioned inside the tank 100. For more detail regarding the electrode structure, see FIG. 13 and the accompanying text below.

As can be seen from the figure, it is preferred that the individual electrode wires 143a and 142a are located in grooves 1103 or 1203 in the side walls (see FIGS. 11 and 12 and accompanying discussion for further detail of the grooves) .

In addition, vertical recesses R are formed in the exterior of the tank 100. Although only one pair of vertical recesses R is shown, it should be understood that another pair exists on the opposing outer side. Vertical electrode buses 142c (and 143c, not shown) extend through the vertical recesses R. The electrode stem portions 142b and 143b connect the electrode portions 142a and 143a to the electrode buses 142c and 143c, and further connect to the individual electrode portions 142a and 143a. The electrode stem portions 142b and 143b pass through the tank walls via the holes H and become the electrode portions 142a and 143a that extend substantially horizontally in the grooves 1103 or 1203 in the interior surface of the tank walls. Leads 142d are connected to bus portions 142c and are further connected to a power source having a negative potential (-) .

Leads 143d are connected to bus portions 143c and are further connected to a power source having a positive potential (+) . The figure shows the electrodes 142 and 143 being located in the front and back walls of the tank 100, but it should be understood that the electrodes could also be located in the end walls. FIG. 10 shows a portion of the electrophoresis tank 100, showing one embodiment of level control devices. The tank 100 may accommodate both a smaller gel slab 110S and a larger gel slab 110L. As can be seen from the figure, the larger gel slab 110L requires a deeper buffer fluid in order to be properly electrophoresed. Therefore, the computer 510 must be able to maintain two different buffer fluid depths.

The initial fill level is set by either the upper outlet 912 or the lower outlet 913, as previously discussed. The upper and lower outlets 912 and 913 set the buffer fluid level during filling of the tank 100 and during insertion of the gel slabs 110. However, the buffer fluid level may drop during the electrophoresis process, leading to a need for additional buffer fluid. Therefore, the computer 510 must be able to detect a low buffer fluid level so that buffer fluid may be added. In addition, the computer 510 must be able to determine a low buffer fluid level for both the larger gel slab 110L and the smaller gel slab 110S.

The tank 100 may therefore include a lower float sensor 1005 and an upper float sensor 1010. The lower float sensor 1005 indicates a smaller gel slab acceptable range 1006, while the upper float sensor 1010 indicates a larger gel slab acceptable range 1008. The float sensors prevent the buffer fluid from becoming too low, and may signal the computer 510 to add buffer fluid. The lower float sensor 1005 is positioned at a lower level in the tank 100 and may be used to detect a low condition when smaller gel slabs are being electrophoresed. Conversely, the upper float sensor 1010 is positioned at a higher level in the tank 100 and may be used to detect a low condition when larger gel slabs are being electrophoresed.

When a float sensor reaches a bottom position, an electrical signal may be passed to the computer 510. A float sensor may also generate a signal when at an upper travel limit (this signal may be used to determine when the tank 100 is full) . Alternatively, a float sensor may generate a position dependent, continuously varying analog signal. The computer 510 may then cause more buffer fluid to be added to the tank 100 (a predetermined volume may be added, with a corresponding upper or lower outlet 912 or 913 removing any excess buffer fluid) .

FIGS. 11 and 12 are cross-sections (enlarged) of first and second embodiments of the vertical tank walls having electrodes passing there through. One or more grooves 1101 or 1201 may be formed on the inner surface of the wall, with the grooves extending substantially horizontally and being spaced apart . The grooves 1101 and 1201 have upper surfaces 1103 and 1203 that are upwardly inclined. The grooves 1101 further include holes H extending through the side wall . Electrode stem portions 143b pass through the holes H, with the holes H being sealed by a silicon sealant or other waterproof sealant. One example is the commercially available silicon sealant known as RTV. The active electrode portion 143a is therefore positioned in the horizontal groove 1101 (see FIG. 9) . The electrode bus 143c extends substantially vertically in the vertical recess R. The active electrode portions 143a include stem portions 143b that connect to the electrode bus 143.

Although the above discussion centers on the electrode 143, the above discussion also applies to the electrode 142. The multiple electrode portions in the grooves 1101 ensure even charge distribution across the tank 100 during electrophoresis .

The angle of the upper surface 1103 or 1203 may range from about twenty degrees to about sixty degrees. The angle of the upper surface 1103 or 1203 causes bubbles, formed during electrophoresis, to travel up and away from the electrode. This is desirable because if bubbles are trapped on or in the near vicinity of an electrode, they can cause problems such as an uneven charge distribution, for example. The grooves 1101 shown in FIG. 11 are formed of generally planar surfaces. In contrast, the grooves 1201 of FIG. 12 are formed of generally curved or non-planar surfaces .

FIG. 13 shows a complete negative electrode 142 and a complete positive electrode 143 according to one embodiment of the present invention. Although each electrode is shown having seven electrode portions 142a and 143a, it should be understood that any number may be employed. The preferred embodiment includes twelve electrode portions 142a and 143a. The electrode portions 142a and 143a extend in the grooves 1103 or 1203 in the inside of the tank 100, while the electrode stem portions 142b and 143b extend through the holes H.

FIG. 14 is a partial cross-section, perspective view of the tank 100 with one wall completely removed and a portion of another wall removed. In addition, the baffles have also been removed (see FIG. 15) . The figure shows the fluid inlets II, 12, 13, and 14 of the re-circulation pumps 804a- 804d. The figure also shows the fluid outlets 01, 02, 03, and 04 of the re-circulation pumps 804a-804d. The inlet II and the outlet 01 communicate with the circulating device 804a (see FIG. 8) , the inlet 12 and the outlet 02 communicate with the circulating device 804b, the inlet 13 and the outlet 03 communicate with the circulating device 804c, and the inlet 14 and the outlet 04 communicate with the circulating device 804d. In addition, the figure shows the cooling tubes 706a, 706b, and 706c, and the receptacles 123 for the sealing strips.

FIG. 15 is similar to FIG. 14 but includes baffles 1501, 1502, and 1503 (described further below with respect to FIGS. 16 and 17) . The baffles may include a plurality of holes 1507 and 1508 for allowing fluid flow. The holes 1507 and 1508 may be formed in any of the baffles.

FIG. 16 is a cross-section of a bottom portion of the electrophoresis tank 100, showing the baffles 1501, 1502, and 1503 (note that the view direction of FIGS. 16 and 17 is opposite to the view direction of FIGS. 14 and 15) . The figure also shows the cooling tubes 706a, 706b, and 706c, the inlets 11-14, the outlets 01-04, and the additional baffle 1609. The baffle 1609 is positioned below the baffle 1502 in order to force the re-circulating fluid around the cooling tubes and around the gel slabs (the fluid flow is illustrated in FIG. 17) . The figure further includes phantom lines that show the installed position of the sealing strips 302. In use, gel slabs 110 are positioned in the plane of the figure, with one end of a gel slab 110 being located in region 101 and the other end being located in region 102.

FIG. 17 uses the cross-section to illustrate the flow of fluid due to the fluid circulating devices. The holes 1507 in the baffle 1502 promote the circulation of fluid between gel slabs in the tank 100. The holes 1507 may therefore be spaced to correspond to gaps between installed gel slabs 110 (see FIG. 15) .

FIG. 18 shows a flowchart 1800 of a computer- implemented method of operating the electrophoresis tank 100. In step 1801 the computer 510 fills the electrophoresis tank 100 with the buffer fluid. The computer 510 may dispense a fixed volume of the buffer fluid, or may fill the electrophoresis tank 100 until the fluid level signal from the fluid level sensor 619 (see FIG. 6) reaches a predetermined value.

In step 1805, the gel slabs 110 are inserted into the electrophoresis tank 100 and between adjacent sealing strips 302. This step is commonly done by a human operator, although it could alternately be performed robotically under the control of a computer. The sealing strips 302 may be put into position before or during insertion of the gel slabs 110.

In step 1808, the buffer fluid level in the electrophoresis tank 100 may be equalized, if necessary. The buffer fluid level may rise during the insertion of the gel slabs 110, and the equalization may need to be performed to remove an excess displaced volume of buffer fluid. Alternatively, the buffer fluid may not be filled to the desired level before gel slab insertion, and may need an additional volume added afterward. The equalization step may therefore entail either addition or removal of buffer fluid.

In step 1812, the computer 510 generates a voltage potential across the electrodes 142 and 143. In a preferred embodiment, about 600 volts (yielding a current of about 5 amps) is placed across the electrodes 142 and 143 for a predetermined electrophoresis time period. The voltage and time variables may of course be varied or chosen in order to achieve a desired electrophoresis result. In a preferred embodiment, the computer 510 controls the applied voltage and current by control of a power supply (see FIG. 5) .

In step 1816, the computer 510 monitors the temperature of the electrophoresis process as one of the important, and controllable, electrophoresis factors. The computer 510 preferably receives continuous or frequent temperature level signals from temperature sensors (see FIG. 7) .

In step 1822, the computer 510 controls cooling devices in order to regulate and stabilize the temperature of the buffer fluid, and indirectly the temperature of the electrophoresis gels 111 of the gel slabs 110 (see FIG. 7) . In step 1827, the computer 510 controls circulating devices to circulate the buffer fluid and therefore ensure even temperature distributions in the first region 101, the second region 102, and the third region 103 (see FIG. 8) . The circulation also ensures that the buffer fluid in the region of the cathode does not suffer from ion depletion, and ensures that the buffer fluid in the region of the anode does not suffer from an ion excess or an ion buildup. Either condition may inhibit current flow and therefore may interfere with the electrophoresis process. In step 1835, the computer 510 may replenish the buffer fluid in the electrophoresis tank 100 in order to prevent ion depletion. A predetermined volume of buffer fluid may be removed at any time during the electrophoresis process and replaced with a substantially equal volume of new buffer fluid.

In step 1837, the computer 510 stops the electrophoresis process. This is done when a predetermined time period has elapsed (the predetermined time period is generally a function of the electrophoresis voltage level) . It should be understood that the computer 510 may also perform additional "wrap-up" duties, such as an additional, optional cool-off phase or a draining of the electrophoresis tank 100.

While the invention has been described in detail above, the invention is not intended to be limited to the specific embodiments as described. It is evident that those skilled in the art may now make numerous uses and modifications of and departures from the specific embodiments described herein without departing from the inventive concepts .

Claims

What is claimed is:

2 1. An electrophoresis tank adapted for use with a

2 plurality of electrophoresis gel slabs, comprising:

3 an outer shell forming an electrophoresis compartment

4 capable of containing a buffer fluid;

5 a plurality of receptacles attached to a bottom of said 5 tank, said plurality of receptacles being adapted to receive 7 a plurality of sealing strips and being formed in two g substantially parallel rows and dividing said tank into a 9 first region, a second region, and a third region, with said 0 third region being disposed between said first region and 2 said second region; and 2 a first electrode at a first side of said tank and a 3 second electrode at a second side of said tank, with said 4 first electrode being entirely within said first region and 5 said second electrode being entirely within said second 5 region; 7 wherein in use a plurality of electrophoresis gel slabs 8 are inserted in between said plurality of sealing strips so 9 that a first end of a gel slab is positioned in said first 0 region and a second end of said gel slab is positioned in i said second region and a main body of said gel slab extends 2 through said third region, and wherein when said tank is 3 filled with said plurality of gel slabs, said plurality of 4 sealing strips contact said plurality of gel slabs to form said third region, with said third region being substantially fluid tight and electrically insulated from said first region, said second region, and said third region.

2. The electrophoresis tank of claim 1, wherein said outer shell comprises glass.

3. The electrophoresis tank of claim 1, wherein said outer shell comprises plastic.

4. The electrophoresis tank of claim 1, wherein said outer shell comprises PLEXIGLAS.

5. The electrophoresis tank of claim 1, wherein said outer shell comprises LUCITE.

6. The electrophoresis tank of claim 1, wherein said outer shell comprises a fluid inlet and a fluid outlet.

7. The electrophoresis tank of claim 1, wherein said first electrode on said first side of said tank comprises a plurality of substantially parallel electrodes vertically spaced apart from one another, and said second electrode on said second side of said tank comprises a plurality of substantially parallel electrodes vertically spaced apart from one another.

8. The electrophoresis tank of claim 1, wherein said first electrode on said first side of said tank comprises twelve substantially parallel electrodes vertically spaced apart from one another, and said second electrode on said second side of said tank comprises twelve substantially parallel electrodes vertically spaced apart from one another .

9. The electrophoresis tank of claim 1, wherein an electrode of said first and second electrodes comprises platinum.

10. The electrophoresis tank of claim 1, wherein a first substantially horizontal groove and a second substantially horizontal groove are formed on inner surfaces of said first and second sides of said tank respectively, with said first electrode extending though said first groove, entering and exiting through holes substantially at each end of said first groove, and with said second electrode extending though said second groove, entering and exiting through holes substantially at each end of said second groove.

11. The electrophoresis tank of claim 10, wherein said first and second groove comprise a plurality of first grooves and a plurality of second grooves.

12. The electrophoresis tank of claim 10, wherein said first and second groove comprise twelve first grooves and twelve second grooves.

13. The electrophoresis tank of claim 10, wherein a groove of said first and second grooves has a sloping upper surface.

14. The electrophoresis tank of claim 10, wherein a groove of said first and second groove has a sloping upper surface at an angle of about twenty to about sixty degrees from horizontal.

15. The electrophoresis tank of claim 10, wherein a groove of said first and second groove has a sloping upper surface at an angle of about forty-five degrees from horizontal.

16. The electrophoresis tank of claim 1, wherein said plurality of sealing strips removably plug into said plurality of receptacles.

17. The electrophoresis tank of claim 1, further comprising at least one cooling device and at least one temperature sensor, said at least one temperature sensor capable of measuring a buffer fluid temperature and said at least one cooling device being capable of cooling said buffer fluid.

18. The electrophoresis tank of claim 17, wherein said at least one cooling device further includes a baffle adapted to direct a buffer fluid flow over said at least one cooling device .

19. The electrophoresis tank of claim 18, wherein said baffle is positioned above said at least one cooling device and includes a plurality of holes adapted to direct an upward buffer fluid flow between said plurality of electrophoresis gel slabs.

20. The electrophoresis tank of claim 17, wherein said at least one cooling device comprises a cooling tube.

21. The electrophoresis tank of claim 17, wherein said at least one temperature sensor comprises a thermocouple.

22. The electrophoresis tank of claim 1, further comprising three cooling devices and three temperature sensors disposed in said first region, said second region, and said third region.

23. The electrophoresis tank of claim 1, further comprising at least one fluid level sensor capable of measuring a buffer fluid level .

24. The electrophoresis tank of claim 23, wherein said at least one fluid level sensor further comprises a lower float sensor positioned in said third region and an upper float sensor positioned in said third region with said upper float sensor determining a larger gel slab fluid low level g and said lower float sensor determining a smaller gel slab

₇ fluid low level.

₁

25. The electrophoresis tank of claim 1, further

₂ comprising three fluid level sensors, said three fluid level

₃ sensors being disposed in said first region, said second

₄ region, and said third region.

26. The electrophoresis tank of claim 1, further

₂ comprising at least one fluid circulating device capable of

₃ circulating said buffer fluid.

₁

27. The electrophoresis tank of claim 1, further

₂ comprising-.

₃ at least one fluid circulating device capable of circulating said buffer fluid;

5 at least one inlet port communicating with said at

6 least one fluid circulating device and admitting said buffer

₇ fluid into said electrophoresis tank; and

_Q at least one outlet port communicating with said at

9 least one fluid circulating device and removing said buffer

₁0 fluid from said electrophoresis tank;

₁ wherein said at least one fluid circulating device ι_? circulates said buffer fluid.

28. The electrophoresis tank of claim 26, wherein said at least one fluid circulating device comprises a motor and an impeller.

29. The electrophoresis tank of claim 26, wherein said at least one fluid circulating device comprises a motor and a pum .

30. The electrophoresis tank of claim 26, further comprising four fluid circulating devices, one fluid circulating device being disposed in said first region, one fluid circulating device being disposed in said second region, and two fluid circulating devices being disposed in said third region.

1 31. The electrophoresis tank of claim 1, wherein said

2 electrophoresis tank further includes:

3 at least one temperature sensor;

4 at least one cooling device;

5 at least one fluid level sensor;

5 at least one fluid circulating device; and 7 a computer in communication with said at least one g temperature sensor, with said at least one cooling device, g with said at least one fluid level sensor, and with said at 0 least one fluid circulating device, said computer being 1 capable of filling said electrophoresis tank with a buffer 2 fluid, equalizing a buffer fluid level within said 3 electrophoresis tank after insertion of a plurality of 4 electrophoresis gel slabs, placing a predetermined voltage 5 potential across said first and second electrodes to start 6 electrophoresis, monitoring a temperature level signal from ₇ said at least one temperature sensor, controlling said at 8 least one cooling device according to a corresponding 9 temperature level signal, circulating said buffer fluid 0 using said at least one fluid circulating device, i replenishing said buffer fluid in a region having a cathode 2 electrode in order to maintain a predetermined ion supply 3 level, and stopping electrophoresis after a predetermined 4 electrophoresis time interval

1 32. An electrophoresis tank adapted for use with a

2 plurality of electrophoresis gel slabs, comprising:

3 an outer shell forming an electrophoresis compartment

4 capable of containing a buffer fluid;

5 a fluid inlet located on said outer shell;

6 at least one fluid outlet located on said outer shell;

₇ a plurality of receptacles attached to a bottom of said g tank, said plurality of receptacles being adapted to receive g a plurality of sealing strips and being formed in two

10 substantially parallel rows and dividing said tank into a

11 first region, a second region, and a third region, with said

12 third region being disposed between said first region and

13 said second region;

14 a first electrode at a first side of said tank and a

15 second electrode at a second side of said tank, with said

15 first electrode being entirely within said first region and

17 said second electrode being entirely within said second

18 region; ig at least one temperature sensor disposed in said

20 electrophoresis tank;

2i at least one cooling device disposed in said

22 electrophoresis tank;

23 at least one fluid level sensor capable of measuring a

2 buffer fluid level; at least one fluid circulating device capable of circulating said buffer fluid; and a computer in communication with said three temperature sensors, said three cooling devices, said fluid level sensor, and said two fluid circulating devices, said computer being capable of filling said electrophoresis tank with a buffer fluid, equalizing a buffer fluid level within said electrophoresis tank after insertion of a plurality of electrophoresis gel slabs, placing a predetermined voltage potential across said first and second electrodes to start electrophoresis, monitoring temperature level signals from said three temperature sensors, controlling said three cooling devices according to a corresponding temperature level signal, circulating said buffer fluid using said two fluid circulating devices, replenishing said buffer fluid in a region having a cathode electrode in order to maintain a predetermined ion supply level, and stopping electrophoresis after a predetermined time interval;

wherein in use a plurality of electrophoresis gel slabs are inserted in between said plurality of sealing strips so that a first end of a gel is positioned in said first region and a second end of said gel slab is positioned in said second region and a main body of said gel extends through said third region, and wherein when said tank is filled with said plurality of gels, said plurality of sealing strips contact said plurality of gels to form said third region, with said third region being substantially fluid tight and electrically insulated from said first region and said second region.

33. The electrophoresis tank of claim 32, wherein said outer shell comprises glass.

34. The electrophoresis tank of claim 32, wherein said outer shell comprises plastic.

35. The electrophoresis tank of claim 32, wherein said outer shell comprises PLEXIGLAS.

36. The electrophoresis tank of claim 32, wherein said outer shell comprises LUCITE.

37. The electrophoresis tank of claim 32, wherein said fluid outlet further comprises an upper outlet and a lower outlet, with said upper outlet setting a larger gel slab fill level and said lower outlet setting a smaller gel slab fill level.

38. The electrophoresis tank of claim 32, wherein said fluid outlet further comprises an upper outlet and a lower outlet, said upper outlet setting a larger gel slab fill level and said lower outlet setting a smaller gel slab fill level, and wherein said lower outlet is blocked in order to use said upper outlet .

39. The electrophoresis tank of claim 32, wherein said first electrode on said first side of said tank comprises a plurality of substantially parallel electrodes vertically spaced apart from one another, and said second electrode on said second side of said tank comprises a plurality of substantially parallel electrodes vertically spaced apart from one another .

40. The electrophoresis tank of claim 32, wherein said first electrode on said first side of said tank comprises twelve substantially parallel electrodes vertically spaced apart from one another, and said second electrode on said second side of said tank comprises twelve substantially parallel electrode vertically spaced apart from one another.

41. The electrophoresis tank of claim 32, wherein an electrode of said first and second electrodes comprises platinum.

42. The electrophoresis tank of claim 32, wherein a first substantially horizontal groove and a second substantially horizontal groove are formed on inner surfaces of said first and second sides of said tank respectively, with said first electrode extending though said first groove, entering and exiting through holes substantially at each end of said first groove, and with said second electrode extending though said second groove, entering and exiting through holes substantially at each end of said second groove.

43. The electrophoresis tank of claim 42, wherein said first and second groove comprise a plurality of first grooves and a plurality of second grooves .

44. The electrophoresis tank of claim 42, wherein said first and second groove comprise twelve first grooves and twelve second grooves.

45. The electrophoresis tank of claim 42, wherein a groove of said first and second groove has a sloping upper surface.

46. The electrophoresis tank of claim 42, wherein a groove of said first and second groove has a sloping upper surface at an angle of about twenty to about sixty degrees from horizontal.

47. The electrophoresis tank of claim 42, wherein a groove of said first and second groove has a sloping upper surface at an angle of about forty-five degrees from horizontal .

48. The electrophoresis tank of claim 32, wherein said at least one temperature sensor comprises three temperature sensors, said three temperature sensors being disposed in said first region, said second region, and said third region.

49. The electrophoresis tank of claim 32, wherein said at least one cooling device further includes a baffle adapted to direct a buffer fluid flow over said at least one cooling device.

50. The electrophoresis tank of claim 49, wherein said baffle is positioned above said at least one cooling device and includes a plurality of holes adapted to direct an upward buffer fluid flow between said plurality of electrophoresis gel slabs.

51. The electrophoresis tank of claim 32, wherein said temperature sensor comprises a thermocouple.

52. The electrophoresis tank of claim 32, wherein at least one cooling device comprises three cooling devices, said three cooling devices being disposed in said first region, said second region, and said third region;

53. The electrophoresis tank of claim 32, wherein said at least one cooling device comprises a cooling tube and a ref igeration unit .

54. The electrophoresis tank of claim 32, wherein said at least one fluid level sensor comprises a lower float sensor positioned in said third region and an upper float sensor positioned in said third region with said upper float sensor determining a larger gel slab fluid low level and said lower float sensor determining a smaller gel slab fluid low level .

55. The electrophoresis tank of claim 32, further comprising three fluid level sensors, said three fluid level sensors being disposed in said first region, said second region, and said third region.

56. The electrophoresis tank of claim 32, further comprising: at least one inlet port communicating with said at least one fluid circulating device and admitting said buffer fluid into said electrophoresis tank; and at least one outlet port communicating with said at least one fluid circulating device and removing said buffer fluid from said electrophoresis tank; wherein said at least one fluid circulating device circulates said buffer fluid.

57. The electrophoresis tank of claim 32, wherein said at least one circulating device comprises a motor and an impeller.

58. The electrophoresis tank of claim 32, wherein said at least one circulating device comprises a motor and a pump .

59. The electrophoresis tank of claim 32, wherein said computer is capable of independently controlling a buffer fluid temperature in said first, second, and third regions

60. An electrophoresis tank adapted for use with a plurality of electrophoresis gel slabs, comprising: an outer shell forming an electrophoresis compartment and including an open top; at least one positively chargeable electrode and at least one negatively chargeable electrode; a lid having an open position and a closed position wherein said lid closes said open top of said electrophoresis compartment; a lid sensor interposed between said lid and said outer shell, said lid sensor capable of detecting said open position of said lid; and a computer in communication with said lid sensor; said computer controlling a voltage and current applied to said at least one positively chargeable electrode and said at least one negatively chargeable electrode to perform electrophoresis; wherein said computer terminates said voltage and said current when said lid sensor detects said open position of said lid.

61. The electrophoresis tank of claim 60, wherein said lid sensor comprises a magnetic switch.

62. The electrophoresis tank of claim 60, wherein said ensor comprises a mechanical switch.

63. The electrophoresis tank of claim 60, wherein said ensor comprises an optical switch.

64. The electrophoresis tank of claim 60, wherein said s affixed to said outer shell by at least one hinge.

65. The electrophoresis tank of claim 60, wherein said s secured to said outer shell by at least one latch.

1 66. A computer- implemented method for automating

2 electrophoresis in an electrophoresis tank, comprising the

3 steps of:

4 filling said electrophoresis tank to a predetermined

5 level with a buffer fluid;

6 equalizing a buffer fluid level within said

₇ electrophoresis tank after insertion of said plurality of

8 electrophoresis gel slabs; g placing a predetermined voltage potential across a 0 first electrode and a second electrode to start 1 electrophoresis; 2 monitoring temperature level signals in each region of 3 said three regions; 4 controlling cooling devices in said each region 5 according to a corresponding temperature level signal; 6 circulating said buffer fluid in said each region; 7 replenishing said buffer fluid in a region having a 8 cathode electrode in order to maintain a predetermined ion 9 supply level; and 0 stopping electrophoresis after a predetermined i electrophoresis time interval.

67. The method of claim 66, wherein said filling step further includes : monitoring a fluid level signal of said electrophoresis tank, with said monitoring being performed under control of said computer; and adding buffer fluid until said fluid level signal reaches a predetermined signal level.

68. The method of claim 66 , wherein said equalizing step includes adding an additional quantity of buffer fluid until said fluid level signal reaches a predetermined signal level .

69. The method of claim 66, wherein said equalizing step includes removing a quantity of buffer fluid until said fluid level signal reaches a predetermined signal level.

70. The method of claim 66, wherein said predetermined voltage potential is about six hundred volts .

71. The method of claim 66 , wherein said controlling step includes increasing a cooling capacity of a cooling device when said corresponding temperature level signal exceeds a predetermined high temperature .

72. The method of claim 66, wherein said controlling step includes increasing a cooling capacity of a cooling device when said corresponding temperature level signal exceeds about twenty degrees Centigrade.

73. The method of claim 66 , wherein said controlling step includes decreasing a cooling capacity of a cooling device when said corresponding temperature level signal is less than a predetermined low temperature.

74. The method of claim 66, wherein said controlling step includes decreasing a cooling capacity of a cooling device when said corresponding temperature level signal is less than about ten degrees Centigrade.

75. The method of claim 66, wherein said circulating step includes circulating said buffer fluid in said each region according to a corresponding temperature level signal.

76. The method of claim 66, wherein said replenishing step further includes : drawing said buffer fluid out of said electrophoresis tank at a predetermined rate; and concurrently adding unused buffer fluid at said predetermined rate .